Studies on the inhibition of RAW264 macrophage cell line chemotaxis by 3-deazaadenosine have led us to postulate that incubation of cells with 3-deazaaadenosine inhibits methylation reaction(s) required for the formation of functional mRNA coding for one or more chemotaxis proteins. Efforts to identify proteins that may play a central role in RAW264 chemotaxis have been limited because chemically defined attractants for RAW264 cells have not been available. This problem was overcome by the isolation of a stable cell hybrid from a fusion between human leukocytes and a thioguanine-resistant RAW264 cell line. The hybrid expressed functional genes for chemotaxis to fMet-leu-phe, a commercially available synthetic attractant. The cell hybrid, granulocytes from several species (mouse, guinea pig, and rabbit) and human monocytes exhibited chemotaxis to fMet-leu-phe and to fMet-leu-phe oxidized to either the sulfoxide or sulfone. In contrast, human neutrophils did not migrate to oxidized fMet-leu-phe. These observations indicated that the human neutrophil may be unique in its lack of chemotactic responsiveness to oxidized fMet-leu-phe, and suggested that the fMet-leu-phe receptor complex or chemotaxis transduction mechanism may be different in human neutrophils than in other phagocytic leukocytes. We have shown that one or more guanine nucleotide binding proteins are required for chemotaxis by RAW264 and the hybrid cells. This conclusion is based on the observation that chemotaxis of either RAW264 or hybrid cells is inhibited upon incubation of the cells with either cholera toxin or pertussis toxin. For both toxins, entry into the cell is required and there is a correlation between toxin-catalyzed ADP-ribosylation of a guanine nucleotide binding protein and the inhibition of chemotaxis. By immunochemical and electrophoretic techniques, the pertussis toxin substrate involved in chemotaxis has been identified as Gi-2, a protein that is also found in brain. In addition, a second family of guanine nucleotide binding proteins of Mr 20-23K has been identified in RAW264 cells. Whether or not this family of proteins is involved in chemotaxis has not yet been determined.